Transcription factors are central to the regulation of pattern formation in development Drosophila segmentation genes, many of which encode transcription factors, participate in a regulatory cascade that ultimately specifies the developmental fates of cells in the embryo. The paired (prd) segmentation gene, the subject of this proposal, functions in combination with other pair-rule genes to regulate the transcription of segment- polarity genes, which are downstream in the cascade. The prd gene is of particular interest because like its mammalian homolog, Pax3 (associated with human Waardenburg's syndrome), it encodes several conserved motifs, including a homeodomain and a paired domain, each of which has DNA binding. activity, and a proline-rich transcriptional activation domain. By analyzing the DNA and protein interactions of the Prd protein using several complementing approaches, this study should provide significant insights into the combinatorial mechanisms by which cell fates are specified during development. The specific aims are: (1) To investigate the requirements for the two DNA-binding domains of Prd. The specificities and relative positions of the paired domain and homeodomain within the Prd protein will be changed, and the resulting constructs will be tested using ectopic expression and rescue assays in the Drosophila embryo. (2) To identify and characterize protein interactors of Prd using the yeast two-hybrid system to screen a Drosophila embryonic cDNA library. Putative interactors will be characterized using in vitro and in vivo assays. (3) To identify nuclear localization signals of Prd using a deletion analysis in Drosophila Schneider cells. Deletion constructs will be tested in vitro for protein interactions with a panel of Prd interactors to identify putative components of the nuclear localization machinery. (4) To assess the importance of the proline-rich activation domain for combinatorial regulation. The functions of Prd chimeras in which the proline-rich domain has been replaced with other classes of activation domain will be tested in embryos. (5) To identify DNA targets of Prd using a reporter gene analysis in embryos. Altered versions of identified targets will be tested to determine the sequence requirements for Prd function. (6) To compare the functions of mouse Pax3 and Drosophila Prd by analyzing the functions of Pax3/Prd chimeras in Drosophila embryos. Evolutionarily- conserved molecular properties of Pax3 that might also be important for Pax3 function in mammalian cells will be analyzed.